Antimicrobial Peptides

by

Dr.RabiaSabir

(MPhil Scholar at The Institute of Microbiology, UAF)

The rise and spread of antibiotic resistance among bacterial pathogens has created a major health care crisis. Among Gram-positive bacteria, the incidence of methicillin-resistant Staphylococcus aureus exceeds 50% in most countries. Vancomycin-resistant enterococci have spread over the last 20 years to become a major cause of nosocomial infections. In Gram-negative pathogens, production of extended spectrum β-lactamasesis reported in 5-8% of Escherichia coli and Klebsiella pneumoniae. Until recently, carbapenem was the antibiotic of choice to treat these infections. However, carbapenem resistance mediated by carbapenemases arose and spread internationally. The therapeutic options to treat these infections are limited to very few antibiotics such as colistin and tigecycline, which are not necessarily optimal therapies because of toxicity or tissue penetration issues. Since there are very few new antibiotics in the drug development pipeline, especially for the treatment of Gram-negative infections, it becomes critical to develop alternatives to classical antibiotics and identify new approaches to treat infectious diseases.

Antimicrobial peptides (AMPs) are critical components of the host innate immune system that serve as “endogenous antibiotics”. Most organisms produce AMPs, including bacteria, fungi, plants, insects and vertebrates. They are multifunctional molecules with antimicrobial activity against bacteria, fungi, viruses and protozoan parasites; they also have numerous immunomodulatory functions. Some AMPs also exhibit antitoxic activity; they neutralize bacterial toxins, including lipid A of lipopolysaccharide (LPS). AMPs are also able to prevent biofilm formation and act on pre-formed biofilms. Mature AMPs are small (<50 amino acid residues), positively charged (+1 to +11) and have amphipathic

properties. They are diverse in amino acid sequence and adopt various secondary structures. In mammals, there are two major groups of AMPs, the α-helical cathelicidins and the β-sheet defensins. Both cathelicidins and defensins are found in large amounts in neutrophil granules and are expressed at mucosal surfaces, where they are synthesized as inactive prepropeptides that are processed into biologically active peptides by host proteases.

Mechanism of action of AMPs:

The mechanisms of action of AMPs are diverse and in some cases specific. Most AMPs appear to exert their bactericidal activity by interacting with the negatively charged bacterial membrane through electrostatic interactions and then forming pores into the cytoplasmic membrane, which leads to bacterial cell lysis.

Functions of AMPs:

AMPs have both direct bactericidal and immunomodulatory activities.In addition to their direct antibacterial activity, AMPs participate in multiple aspects of immunity. In respect to their immunological functions, AMPs are also known as host-defense peptides. By interacting with a variety of host cell receptors, AMPs promote therecruitment of leukocytes to the site of infection through both direct chemotactic activity and stimulation of chemokine production by leukocytes, epithelial cells and other cell types. AMPs also modulate host responses to microbial compounds, inducing both pro- and anti-inflammatory signals; for example LL-37 inhibits TNFα production and other host responses to LPS. AMPs can impact the adaptive immune response by influencing antigen presentation, cell recruitment, and by modulating B- and T-cell responses. Finally, some AMPs also play a role in angiogenesis and wound healing.

Bacterial resistance to host AMPs:

During the co-evolution of hosts and microbes, bacteria have developed several strategies to resist and survive the activities of AMPs. Both Gram-positive and negative bacteria are able to sense the presence of AMPs in the environment through two-component regulatory systems. For example, Salmonella entericaand Streptococcus pyogenesuse the PhoPQ and CsrRS two-component systems, respectively, to sense LL-37. Activation of these signaling pathways by AMPs results in the upregulation of genes associated with AMP resistance. These AMP resistance mechanisms can be grouped according to their mode of action into shielding structures, proteases, surface charge alterations, ABC transporters, and modulation of AMP gene expression.

1. Shielding of the bacterial cell surface Surface structures external to the bacterial cell envelope, such as capsule polysaccharides (CPS), play an important role in AMP resistance. They are proposed to act as a protective shield binding AMPs and reducing the amount of AMPs that reaches the bacterial membrane. Specifically, anionic CPS of both Gram-positive and Gram-negative bacteria binds cationic AMPs to promote resistance. CPS are not the only bacterial shielding structures, curli fimbriae from uropathogenicEscherichia coli and the M1 protein from group A Streptococci bind LL-37 to promote resistance.

2. Proteolytic degradation of AMPs Both Gram-positive and Gram-negative bacteria produce membrane-bound and/or secreted proteases that can degrade and inactivate AMPs. For example, the outer-membrane protease OmpT of enterohemorrhagicE. coli cleaves and inactivates the α-helical LL-37 but cannot cleave HNP-1, which is stabilized by disulfide bonds. Secreted proteases, such as the Zn++-metalloproteaseZapA from Proteus mirabilis degrades LL-37 and hBD-1, whereas aureolysin from S. aureusinactivates LL-37, indicating specific substrate preference for AMPs.

3. Charge alterations Bacteria minimize their interaction with AMPs by reducing the net negative charge of their membrane. Due to the basic physiological differences in the composition of the Gram-positive and Gram-negative bacterial cell walls, they use different mechanisms to reduce their net negative charge. In Gram-positive bacteria, the net negative charge of the cell wall is decreased by the addition of D-alanine to the phosphates of teichoic acids using a process that occurs nearly ubiquitously among species. In Gram-negative bacteria, the phosphate groups present on the lipid A and core moieties of LPS are responsible for the netnegative charge of the outer-membrane. To neutralize the net negative charge of LPS, the lipid A and core are covalently modified with positively charged moieties that mask the phosphate groups, thereby preventing AMP binding.

4. ABC transporters ABC transporters are general transport systems that are used to import or

export a variety of substrates across the membranes of both Gram-positive and Gram-negative bacteria. In Gram-positive bacteria, these transporters have developed a unique relationship with two-component signal transduction systems to promote greater AMP resistance. In Gram-negative bacteria, the import and/or export activities of ABC transporters are used by various species to resist killing by AMPs. For example, the MtrC export-type transporter is used by Neisseria gonorrhoeaeand Haemophilusducreyito resist hBDs and LL-37, whereas the SapA import-type transporter is used by Haemophilusinfluenzaeto deliver AMPs into the cytosol where they are degraded by proteases and recycled as nutrients.

5. Down regulation of AMP gene expression Some pathogens actively suppress expression of AMP genes by host cells. For example, Shigellaflexneriinhibits the expression of LL- 37 in intestinal epithelial

cells. Similarly, Helicobacter pylori selectively inhibits the expression of hBD-3, which is particularly active against Helicobacter pylori, through a mechanism involving the virulence factor CagA that interferes with cell signaling upon

translocation into host cells.

Current Clinical Use of AMPs The production of AMPs is not limited to multicellular organisms; bacteria can also synthesize AMPs that are active against other bacteria. These AMPs of bacterial origin that consist of non-ribosomally synthesized peptides such as polymyxins, bacitracin and gramicidins and ribosomally synthesized peptides such as bacteriocins, have also been used. Polymyxin E (also known as colistin) is a cyclic lipopeptide produced by Bacillus polymixa. Since 1959, polymyxin E has been used for the treatment of infections caused by Gram-negative bacteria. It was replaced by aminoglycosides in the 1980s, because of concern about toxicity. Polymyxin E has reemerged over the last 15 years and is currently one of the last-resort drugs for treatment of multi-drug resistant Enterobacteriaceae, Pseudomonas spp. and Acinetobacterspp.Gramicidin S, a cyclic decapeptide produced by Bacillus brevis, has been used as a topical antibiotic against Gram-

positive bacteria. Nisin is a bacteriocin produced by Lactococcuslactisthat acts primarily against Gram-positive bacteria and has been used safely as a food preservative.Another application is acne treatment. Acne lesions by Propionibacterium acnes are associated with moderate-to-severe inflammation; atreatment involving both antimicrobial and anti-inflammatory agents would be of significant benefit.Thatcould be improved by an indolicidin-derived peptide named MBI-594AN from Migenix. This peptide is not only antimicrobial against P. acnes, but also suppresses P. acnes-stimulated cytokine release. Thus AMPs have now been used for therapeutic purpose due to antibiotic resistant problem.